大鼠肝tRNA~(Ile)基因的克隆与体外转录

采用PCR扩增出大鼠肝tRNAIle基因,构建了重组质粒pGWIW,并用T7RNA聚合酶／启动子系统对其进行了体外表达．经过对转录产物片段大小及运用Northernblot鉴定,证明获得了不含修饰核苷酸的大鼠肝tRNAIle生物学活性检测显示：合成基因体外转录产物氨基酰化活性是天然tRNA的40％,提示修饰核苷酸在哺乳动物Ile－tRNA合成酶的识别过程中起重要作用     

用T7 RNA聚合酶体外转录合成大鼠肝tRNAIle

采用PCR技术从rec-M13mp18中扩增出120 bp的大鼠肝tRNA^Ile合成基因片段,经限制性内切酶BstNⅠ酶切后作为模板,利用T7 RNA聚合酶在体外无细胞体系转录由T7启动子带动的大鼠肝tRNA^Ile基因,生成不含修饰碱基的tRNA^Ile,并对体外转录反应条件进行了优化,回收的tRNA产量可达DNA模板量的40倍.     

肝癌细胞AFP基因高水平的转录是受核蛋白成分的促进

利用大鼠甲胎蛋白(AFP)基因片段作为模板,分析大鼠肝癌细胞核蛋白成分对体外转录活性的影响,发现大鼠肝癌含有促进AFP基因体外转录的核蛋白。作为对照．没有发现任何成年大鼠肝核蛋白可以促进AFP基因的体外转录。为了确定促进AFP基因体外转录的核蛋白作用部位,对AFP基因模板5＇端上游序列进行了不同程度的删除,进一步分析核蛋白对删掉5’端上游序列后的模板体外转录的影响,结果表明,AFP基因转录的起始点到255bp这段DNA序列是大鼠肝癌核蛋白促进AFP基因转录必不可少的。以SV40DNA经Pst I酶酶切所得的DNA片段(1216bp和4027bp)代替AFP基因片段作为模板,不存在核蛋白促进体外转录的现象。用AFP基因转录的起始点到 255bp这段的DNA为探针,进行Southwestm印迹分析,结果发现了8种与探针结合的核蛋白。     

斑马鱼Gfi1.1基因的克隆及其体外转录

目的:克隆斑马鱼Gfi1.1基因的全长cDNA,运用T7 RNA聚合酶对含有Gfi1.1基因的ORF区进行体外转录,在体外合成5端带有帽子结构的Gfi1.1 mRNA分子,为后续研究斑马鱼Gfi1.1基因的功能打下基础。方法:应用RT-PCR从斑马鱼组织中扩增出Gfi1.1 cDNA片段,经回收纯化与pGM-T载体连接并转化感受态细菌DH-5α,通过蓝白筛选酶切鉴定阳性菌落,小量提取质粒,Nde I限制性内切酶线性化pGM-T-Gfi1.1质粒,运用T7 RNA聚合酶对Gfi1.1基因进行体外转录及加帽。经凝胶电泳对目的片段进行鉴定。结果:RT-PCR扩增获得约1.2 kb的DNA片段,DNA序列分析的结果与GenBank上的序列(NM_001020776)一致,酶切线性化及体外转录加帽pGM-T-Gfi1.1,凝胶电泳鉴定RNA分子大小与预期完全一致。结论:成功克隆斑马鱼Gfi1.1基因并体外转录及加帽pGM-T-Gfi1.1。     

大肠杆菌等受体tRNALeu的T7 RNA聚合酶转录

用化学方法合成编码2个大肠杆菌tRNALeu(tRNALeu1和tRNALeu2)的基因和T7启动子,分别克隆到pUC19载体上,并在纯化的T7 RNA聚合酶的体外转录系统中转录出不含修饰核苷酸的tRNALeu.在T7转录体系中,亚精胺对转录有负影响.在最适转录条件下,可以得到有活力的RNA转录物的量是模板DNA的250倍左右.在大肠杆菌亮氨酰-tRNA合成酶的催化下,2种经体外转录产生的未修饰等受体tRNALeu(tRNALeu1和tRNALeu2)的亮氨酸接受能力基本相同,但只有从体内纯化对应的tRNALeu的四分之一左右,表明修饰核苷酸在tRNALeu氨酰化过程中起着较为重要但非关键的作用.     

rmf基因启动子识别因子选择性研究

分别用σ^(70)或σ^(38)因子和核心酶(Core enzyme)重建RNA聚合酶全酶(Holoenzyme)对含有rmf基因启动子的DNA模板进行体外转录。不同的限制性内切酶酶切片段模板证实了rmf基因转录起始位点,尽管rmf基因在大肠杆菌进入稳定期大量表达,但是其启动子对稳定期起重要作用的σ^(38)因子的识别能力很低,而只能被σ^(70)所识别。rmf基因启动子体外转录的最适温度为37℃,最适NaCl浓度为50mmol/L。     

利用PCR技术构建体外高效转录系统

设计并合成了一对 PCR 反应引物,其5′端引物除含有目的基因5′端序列外,还外加 T7 RNA 聚合酶启动子的17个核苷酸.3′端引物则按常规设计.以染色体 DNA为模板,通过 PCR,可扩增出带有 T7 RNA 聚合酶启动子的目的基因 DNA 片段.以此 PCR 产物为模板,在体外成功实现了高效转录.这是一种快速、简便构建体外高效转录系统的好方法.     

siRNA抑制丙型肝炎病毒IRES介导的基因表达

以HCVIRES为靶位 ,应用T7RNA多聚酶体外转录合成了 5条小干扰RNA(siRNA) .脂质体转染法将其导入HCVIRES介导萤光素酶表达的转基因细胞 (HepG2 .970 6 )中 ,通过测定萤光素酶的量 ,评价T7siRNA对HCV介导基因表达的抑制作用 .结果表明 ,所合成的 5条T7siRNAs ,均能特异性地抑制萤光素酶基因的表达 ,抑制率分别为 94 31%、80 0 1%、78 0 1%、80 33%、85 6 4% ,其中以靶向HCVIRES第二茎环结构的T7siRNA1抑制率最高 ,且对HCV基因的抑制作用有剂量依赖性 ,随T7siRNA1量的增加 ,抑制率逐渐增强 .siRNA抑制HCV基因的作用具有良好的特异性 ,改变其中 1个核苷酸即无显著抑制作用 .     

Mip1对大鼠心肌细胞凋亡基因Bid转录调控作用的初探

目的:研究转录因子Mip1对大鼠心肌细胞H9C2凋亡相关基因Bid的转录调控作用.方法:以H9C2细胞基因组DNA为模板,扩增Bid核心启动子片段,将其克隆入荧光素酶报告基因质粒PGL3-Basic中,构建重组载体,并采用双酶切法、PCR法及基因测序对其进行鉴定.用脂质体转染法将该载体转入Mip1不同程度过表达的H9C2细胞,检测该细胞Bid基因启动子区的转录活性.结果:成功克隆Bid基因启动子区,双酶切、PCR和基因测序均显示PGL3-Basic-Bid promoter重组载体构建成功.荧光素酶相对活性检测显示在H9C2细胞中,随着Mip1转入的增多,Bid启动子区转录活性逐渐下降.结论:Mip1作为一个新的转录抑制因子,可以下调凋亡基因Bid的转录.     

银松素合酶基因地高辛标记RNA探针的合成

目的:体外转录合成银松素合酶(PS)基因地高辛标记反义RNA探针。方法:通过双酶切把PScDNA全长序列接到pBlueskriptⅡKS( )的多克隆位点上;同时,经由NCBI比对确定PScDNA的特异序列,设计含有SalⅠ和XbaⅠ这2个酶切位点的上、下游引物,用PCR法扩增含有该位点的目的片段,并连接到pBlueskriptⅡKS( )的多克隆位点上;最后利用pBlueskriptⅡKS( )上的启动子T7,用T7RNA聚合酶在体外转录合成地高辛标记PS全长及特异序列的反义RNA探针。结果:通过对构建探针的单、双酶切,PCR及电泳鉴定,表明成功合成了PS基因地高辛标记的全长及特异序列的反义RNA探针。结论:RNA探针的合成为后续马尾松PS基因表达的RNA原位杂交研究打下了基础。     

Materials for Chinese Phyllanthoideae

The present paper is part of taxonomic study on Chinese Phyllanthoideae. Included in it are two new varieties, Leptopus esquirolii var. villosus and Drypetes hainanensis var. longistipitata, one new combination, Glochidion triandrum var. siamense, and seven new records in China: Drypetes salicifolia, D. hoaensis. Actephila subsessilis, Glochidion khasicum, G. nubigennum, Bridelia spiosa and B. poilanei. In addition, seventeen taxon names are newly reduced: Liodendron formosanum = Drypetes formosana, Liodendron matsumurae = Drypetes matsumurae, D. longipes = D. indica, Antidesma paxii = A. acidum, A. hiiranense, A. filipes and A. pentandrum var. hiiranense = A. japonicum, A. calvescens = A. montanum, A. microphyllum = A. venosum, Breynia stipitata var. formosana and B. jormosana = B. vitis-idaea, Glochidion zeylanicum var. tomentosum = G. hirsutum, G. rubidulum = G. thomsonii, G. acuminatum = G. triandrum, G. fagifolium and Phyllanthus fagifolius = Glochidion sphaerogynum, Bridelia penangiena = B. insulana, B. henryana = B. tomentosa. All the types are kept in SCBl and PE.     

Phylogenetic revision of the Taenogera Kröber genus-group (Diptera: Therevidae), with descriptions of two new genera

The phylogenetic relationships of the Taenogera genus-group, consisting of nine genera, are examined using cladistic methods. Twenty-one species representing nine genera and one outgroup were compared in a cladistic analysis across 99 states in 44 characters. The genus Taenogera Kröber is revised to contain only T. longa (Schiner), T. nitida (Macquart) and T. notatithorax Mann. Two new genera are described and figured: Actenomeros gen. n., with two species, A. corniculaticaudus sp. n. and A. onyx sp. n.; and Taenogerella gen. n., with four species, Ta. elizabethae sp. n., Ta. platina sp. n., Ta. schlingeri sp. n. and Ta . nigrapicalis (Mann) comb. n. Phylogenetic analysis also supports the removal of Nanexila gracilis (Mann) comb. n. from Taenogera .     

Relationships between host blood factors and proteases in Glossina morsitans subspecies infected with Trypanosoma congolense

Abstract. Host blood effects on Trypanosoma congolense establishment in Glossina morsitans morsitans and Glossina morsitans centralis were investigated using goat, rabbit, cow and rhinoceros blood. Meals containing goat erythrocytes facilitated infection in G. m. morsitans , whereas meals containing goat plasma facilitated infection in G. m. centralis. Goat blood effects were not observed in the presence of complementary rabbit blood components. N-acetyl-glucosamine (a midguMectin inhibitor) increased infection rates in some, but not all, blood manipulations. Cholesterol increased infection rates in G. m. centralis only. Both compounds together added to cow blood produced superinfection in G. m. centralis , but not in G. m. morsitans. Midgut protease levels did not differ 6 days post-infection in flies maintaining infections versus flies clearing infections. Protease levels were weakly correlated with patterns of infection, but only in G. m. morsitans. These results suggest that physiological mechanisms responsible for variation in infection rates are only superficially similar in these closely-related tsetse.     

Selection of Verticillium lecanii Isolates with High Potential for Biocontrol of Cucumber Powdery Mildew by Means of Components Analysis at Different Humidity Regimes

To identify characteristics for the selection of Verticillium lecanii isolates with high potential for biocontrol of Sphaerotheca fuliginea under glasshouse conditions, an exploratory study was performed on the effect of water limitation on the development of 14 isolates. The conidial germination, growth and sporulation of isolates of V. lecanii were studied in a tritrophic system on cucumber leaves and in a ditrophic system in Petri dishes. Their mycoparasitic ability was studied in S. fuliginea and Cladosporium cladosporioides . All characteristics were clearly affected by humidity. Four isolates showed good biocontrol potential. The performance of isolates on agar had less predictive value than on powdery mildew. The germination of isolates of V. lecanii was lower and the mycelial growth faster on agar than on mildewed leaves under corresponding humidity conditions. The results suggest that conditions in the phyllosphere differed from the set humidity in the surrounding air. A correlation was found between the lysis of C. cladosporioides growing in dual culture on agar with isolates of V. lecanii and the parasitism of powdery mildew on detached, rooted leaves. C. cladosporioides might offer a suitable substrate for testing isolates of V. lecanii for mycoparasitic potential under various environmental conditions. Conidial germination, growth and sporulation had limited predictive value.     

Evaluation of the Palaearctic Eurithia consobrina (Diptera: Tachinidae) as a Potential Biocontrol Agent for Mamestra configurata (Lepidoptera: Noctuidae) in Canada

The evaluation of Eurithia consobrina (Meigen) as a candidate for introduction against bertha armyworm, Mamestra configurata (Walker) (Lepidoptera: Noctuidae), in western Canada included studies of its host range, distribution, synchronization with host, constancy, abundance and life history in Europe. In addition, its diapause induction and its coldhardiness were compared with those of the target host M. configurata and its native tachinid parasitoid, Athrycia cinerea (Coq.). E. consobrina was found to fit criteria for a successful biocontrol agent reasonably well. It also had the potential to fill a largely unoccupied niche in the parasitoid complex of M. configurata. E. consobrina has a facultative diapause of the long-day type, similar to A. cinerea, but is less sensitive to diapause-inducing conditions. E. consobrina is at least as coldhardy as A. cinerea and more coldhardy than M. configurata. Laboratory exposures to temperatures of 0 C for 140 days apparently selected for greater coldhardiness, and later generations survived exposures of 140 days at -15 C without significant mortality. Adult E. consobrina were released at three locations in Manitoba in 1986 and 1987, but establishment has not been confirmed.     

Predation by Halmus chalybeus (Coleoptera: Coccinellidae) on Ceroplastes destructor and C. sinensis (Hemiptera: Coccidae: Ceroplastinae) Infesting Citrus in Northland, New Zealand

Predation by Halmus chalybeus (steelblue ladybird) on two species of wax scale was studied on citrus orchards in Northland, New Zealand. Field experiments using an exclusion technique of enclosing citrus branches in bags, found that larval and adult H. chalybeus preyed on first and second instars of both Ceroplastes destructor (white wax scale) and C. sinensis (Chinese wax scale), but not third instar C. destructor. Third instar C. sinensis and adults of both species were not tested but are rarely, if ever, attacked by H. chalybeus. The sampling of scale and ladybird populations and field experiments showed that few scales of either species survived past the second instar stage where H. chalybeus was numerous. The feeding rate of H. chalybeus on settled first and second instar scales was estimated inside bagged branches. Adults consumed on average 15.6 C. destructor and 13.3 C. sinensis per day per ladybird, while larvae ate 9.7 C. destructor per day. These feeding rates accounted for the experimental reduction of scale populations. H. chalybeus is a useful natural enemy for the control of C. destructor and C. sinensis when its activity is not disrupted by pesticides.     

Further flavonoid studies onAttalea species and some related cocosoid palms

In a flavonoid survey of direct and hydrolysed leaf extracts of sixteenAttalea, sevenScheelea and fourOrbignya species free tricin, tricin 7-glycosides, tricin 5-glucoside and flavone C-glycosides were the most frequent constituents; present in 100, 89, 70, and 81% of species, respectively. Luteolin, quercetin and isorhamnetin were each found in only 15% of the sample. The present results confirm the findings of a previous survey thatAttalea, Scheelea andOrbignya are chemically heterogeneous with as much variation between species as between genera. Furthermore, threeAttalea species,A. allenii, A. guaranitica andA. victoriana showed some infraspecific variation. On the other hand all three accessions ofA. ferruginea and six ofA. geraensis examined gave identical flavonoid profiles. The results support the view thatA. geraensis andA. guaranitica are closely related but do not support the suggested close relationship based on morphology betweenA. oleifera, A. burretiana andA. piassabossu.     

Investigations into the evolutionary history of the polyploid complexAntennaria neodioica (Asteraceae: Inuleae)

TheAntennaria neodioica polyploid agamic complex is a polymorphic species occurring across North America mainly north of the terminal margin of the Wisconsin glacier. This taxonomically difficult group has recently been treated as consisting of the four subspeciesA. neodioica subsp.canadensis, subsp.howellii, subsp.neodioica, and subsp.petaloidea. TheA. neodioica agamic complex has been considered of hybrid origin with several sexual diploid species constituting its parentage. Crosses were made among five sexual diploid species ofAntennaria, morphologically similar toA. neodioica s.l., in an attempt to discover its origins. Representative specimens of the five diploid species,A. neodioica s. l., and the synthetic interspecific F₁ hybrids were subjected to various analyses including PCA, cluster (UPGMA), and discriminant analyses. Results suggest that theA. neodioica complex is of multiple hybrid origin involving the four diploid speciesA. neglecta, A. plantaginifolia, A. racemosa, andA. virginica. BecauseA. neodioica is the result of diverse origin it is more desirable to consider the agamic complex as a separate, distinct species from its sexual diploid relatives. Several morphological characters in the diploid species were determined to be polygenically inherited.Investigations into the Evolutionary History of the Polyploid Complexes inAntennaria (Asteraceae: Inuleae). I.